The observations indicated that the increased root biomass phenotype in the activation lines was due to the elevated OsZHD2 expression levels. In addition to the root phenotype, the OX plants and the T-DNA activation line influenced leaf development, so that abaxially curled leaves were observed . The OsZHD2-OX plants exhibited markedly increased root development at 14 DAG . Fresh and dry weights of roots were higher for the transgenic lines than for the out segregated WT . To examine whether the increase in biomass improved nutrient uptake, we analyzed the rate at which N was absorbed from a liquid growth medium containing KNO3. Based on the amount of residual N in the medium, the N concentration reduced rapidly and at a higher rate in OX plants than in the WT plants . The results suggested that the former had a higher N uptake capacity via the roots, which would also imply that the OX plants had a higher capacity to tolerate low-N conditions. To test the hypothesis, we grew the plants under low-N conditions in a growth chamber . In mature plants at the booting stage, the N concentration was 1.5- fold higher in the flag leaves of OsZHD2-OX compared with the flag leaves of the WT . The Pi accumulation rate was also 1.5-fold higher in the flag leaves of OsZHD2-OX than in the WT plants . Seed fertility was markedly higher in the over expression plants. Although <30% of the WT seeds were fertile, >50% of the grains from the OX plants were fertile . The results indicated that the uptake of nutrients increased in OsZHD2-overexpressing plants.
Plants were grown in a paddy field under normal N supply. There were no obvious phenotypic differences between the over expression plants and the WT up to maturity. Their architectures were almost identical,growing blueberries including plant height, panicle length, total spikelet number, and fertile seed number . However, the 100-grain weight was higher in the OsZHD2 OX lines . The increase in seed weight was potentially due to increased N uptake.Lateral roots began to emerge from both the WT and the activation lines at 3 DAG. By 4 DAG, the WT laterals were ~0.5 cm long, while those of the activation line were slightly longer . The difference in lengths became more pronounced as the plants grew . We performed transcriptome analyses using mRNA prepared from the total root samples of WT and OsZHD2-D plants at 4 DAG and at 6 DAG . At 4 DAG, 68 genes were up-regulated and 384 genes were down-regulated at least 2-fold in OsZHD2-D . At 6 DAG, 513 genes were up-regulated and 524 were down-regulated at least 2-fold in OsZHD2-D plants . At both stages, 22 transcripts were commonly up-regulated while 54 transcripts were down-regulated at least 2-fold . To verify the RNA sequencing data, we selected four genes that were up-regulated at both stages, in addition to CYCD4;1 and ERF3, which increased only at 6 DAG, and ABCC7 and PUB64, which were down-regulated at both stages . qRT-PCR analyses revealed that their expression patterns were similar to the patterns observed in the results of our RNA sequencing analyses . The findings suggested that the sequence data were reliable.The 22 genes that were up-regulated at both 4 and 6 DAG included two associated with ethylene biosynthesis, S-adenosylmethionine synthetase 2 and ACC oxidase 2 , which suggested that ethylene influenced the root phenotypes .
Ethylene biosynthesis begins with the conversion of methionine to S-adenosylmethione by S-adenosylmethione synthetase, with ATP as a co-substrate . In the following step, ACC is formed from S-adenosylmethione by ACC synthase . The final step is the synthesis of ethylene from ACC by ACC oxidase . Our qRT-PCR assay confirmed that the expression of OsSAM2 and OsACO2 indeed increased in OsZHD2-D lateral roots at both stages . Genes encoding ACS were not placed on the list of induced genes because the differences in transcript levels between WT plants and transgenic plants were <2-fold. However, qRT-PCR analyses revealed that ACS5 transcript levels increased in OsZHD2-D at both stages . Ethylene production measurements from 8 DAG plants showed that OsZHD2-D samples accumulated more ethylene in their roots , shoots , and the whole plant when compared with the WT plants. To examine whether OsZHD2 binds directly to ethylene biosynthesis genes, we performed ChIP assays using transgenic plants over expressing OsZHD2-Myc. Promoter regions P3, P4, and P5 of ACS5 chromatin were enriched by Myc antibodies .To investigate whether the accumulation of ethylene was the major factor responsible for the OsZHD2-D seedling root phenotypes, we investigated the effects of an ethylene biosynthesis inhibitor AVG which reduces ethylene production by blocking ACS activity . The addition of 3 µM AVG reduced lateral root growth in WT plants and rescued the enhanced lateral root growth phenotypes of OsZHD2-D . However, low concentrations of AVG did not affect the lateral growth of OsZHD2-D as well as that of the WT . To examine whether the restoration was due to decreased meristem activity, we performed EdU labeling. The results of the experiment demonstrated that the application of AVG reduced the root meristem activity of the WT and OsZHD2-D significantly .
The results suggest that OsZHD2 enhances meristem activity in the apical region of roots by inducing ethylene accumulation.To determine whether exogenous ethylene treatment promotes root development, 3 DAG seedlings were transferred to MS medium containing various concentrations of ACC. Lateral root length increased significantly when plants were supplied with 10 nM ACC . Previously reported results have suggested that ethylene induces auxin biosynthesis by stimulating the expression of Rice Anthranilate Synthase Alpha-subunit, which encodes an enzyme producing anthranilate, a precursor of Trp . To investigate whether ethylene increases auxin concentrations in rice, we generated transgenic DR5::GUS plants expressing the GUS gene under the synthetic auxin-responsive promoter and their lateral roots exhibited weak GUS expression at the tips and in the basal regions . When plants were exposed to 10 nM ACC, GUS activity was higher in the treated roots than in the control plants grown in the absence of ACC . Staining was also observed in the area between the tips and basal regions where GUS activity had not been observed prior to treatment with ACC. Consistent with the GUS assay results, the GUS transcript levels increased in ACC-treated roots . We also used a DR5::VENUS plant that expressed the yellow fluorescent protein under the influence of the DR5 promoter . The treatment of the plants with ACC increased VENUS signal in the tips and the central stele of the lateral roots . The results of such experiments suggest that ethylene induced auxin biosynthesis in the RAM. The results of qRT-PCR analyses revealed that 10 nM ACC induced the expression of OASA2 as well as auxin biosynthesis genes, TAR2 and YUCCA7, with peaks observed 6 h after treatment . According to the observations, a low concentration of ethylene could induce auxin biosynthesis in rice lateral roots. In Arabidopsis,square plant pot ethylene enhances auxin biosynthesis by increasing the expression of WEI2/ASA1 and WEI7/ASB1, two genes encoding AS sub-units . In rice, OASA1 and OASA2 encode the AS α-subunit . According to the RNA-Seq assay results, OASA2 expression was higher in OsZHD2-D roots , which was validated using qRT-PCR analyses . The major IAA biosynthesis route is the IPyA pathway, which is mediated by TAA/TARs and YUCCA in Arabidopsis . Our RT-PCR results showed that TAR2 was induced in OsZHD2-D roots . We also observed that YUCCA7 expression was higher in the activation line . An analysis of the DR5::GUS plants showed that the expression levels of the GUS reporter were significantly higher in OsZHD2-D roots . All the findings above suggested that OsZHD2 induced IAA biosynthesis. Notably, strong staining was observed in the proximal area of the root tips of OsZHD2-D, which also indicated that OsZHD2 promoted auxin accumulation in the growing region.
To investigate whether AVG treatment affects DR5::GUS expression in OsZHD2-D, we applied 3 µM AVG to the DR5::GUS plants in the WT and OsZHD2-D background. Visualization of GUS expression showed that the reporter expression was decreased by AVG in both plants . This observation supports that the increased auxin biosynthesis in OsZHD2-D was due to elevated ethylene levels. To examine whether OsZHD2 binds directly to auxin biosynthesis genes, we performed ChIP assays using transgenic plants over expressing OsZHD2-Myc. However, we were unable to observe any significant binding of OsZHD2 to the promoter regions of TAR2 and YUCCA7 . To confirm OsZHD2-D phenotypes, we analyzed expression patterns of ethylene and auxin biosynthesis genes in OsZHD2-overexpressing plants. The results of qRT-PCR analyses revealed that expression levels of ethylene and auxin biosynthesis genes are increased in OsZHD2-overexpressing plants .To further study the functional role of OsZHD2, we generated oszhd2 null mutants using the CRISPR/Cas9 [clustered regularly inter spaced short palindromic repeats / CRISPR-associated protein 9] system . Analyses of two independently obtained bi-allelic oszhd2 mutants revealed that the lengths of their seminal roots and lateral roots did not vary considerably from those of the WT and heterozygous plants. The lack of obvious phenotypic changes was potentially due to genetic redundancy. OsZHD2 encodes ZF-HDs, a protein group that includes 11 members in rice . Among them, OsZHD2 is the most homologous to OsZHD1, with 80% identity and 84% similarity at the amino acid sequence level. Plants that over express OsZHD1 exhibit an abaxially curled and drooping leaf phenotype similar to that observed in OsZHD2-OX plants. We isolated a T-DNA tagging line in which T-DNA was inserted 136 bp upstream of the start ATG codon . The expression of OsZHD1 was reduced significantly in the tagging line . For the mutant, no obvious alteration was observed in the phenotype .Since oszhd1 and oszhd2 single mutants exhibited normal root growth, we generated oszhd1 oszhd2 double mutants using the CRISPR/Cas9 system to target the conserved sequence . In the double mutants, lateral root development diminished significantly , indicating that OsZHD1 and OsZHD2 redundantly play roles in the regulation of such development. The transcript levels of SAM2, ACS5, ACO2, OASA2, TAR2, and YUCCA7 also decreased in the oszhd1 oszhd2 double mutants , supporting our hypothesis that the OsZHD genes are involved in the control of the biosynthesis of ethylene and auxin. To observe whether exogenous ethylene treatment would stimulate lateral root development in oszhd1 oszhd2 double mutants, seedlings were grown on N6 medium with or without 1 µM ACC . In the ACC-treated plants, the lengths of the lateral roots of oszhd1 oszhd2 double mutants increased more than the lengths of the lateral roots of the WT plants . These results indicate that the changes in the root architecture observed in oszhd1 oszhd2 double mutants are at least in part due to the defective ethylene biosynthesis.The over expression of OsZHD2 increased ethylene levels and enhanced the expression of genes linked to its biosynthesis. The OsZHD2 transcript is preferentially present in the meristem regions where ACS5 is expressed . Therefore, the primary role of OsZHD2 in root development appears to be the induction of ethylene production by inducing ACS5 expression. Although ethylene generally functions as a growth inhibitor, it occasionally promotes growth, particularly in semi-aquatic plants . Leaf, stem, and root development can be positively regulated by ethylene at relatively low concentrations . In addition, ethylene induces lateral root initiation near the growing root tip and promotes the emergence of lateral root primordia . The overproduction of ethylene through the application of exogenous ACC inhibits lateral root initiation but induces outgrowth of already existing primordia . These observations reported in previous studies support our hypothesis that OsZHD2 enhances root growth by increasing ethylene production in the root tips.Using plants expressing the GUS or VENUS markers under the influence of the DR5 promoter, we showed that a low concentration of ACC induced auxin accumulation in the growing region of lateral roots. We also demonstrated that ethylene increases the expression of auxin biosynthesis genes, including OASA2, TAR2, and YUCCA7 . Expression of the marker genes was promoted strongly in the region near the root tips of OsZHD2-D plants . The above expression trend was similar to that for ACC-induced GUS activity .